Several aspects of contemporary molecular genetics and biotechnology make it desirable to be able to produce genetically-altered proteins. For example, mutated protein domains are sometimes hyper-immunogenic, facilitating the production of neutralizing antibody-based vaccines. Moreover, site-directed mutations, ideally one amino acid at a time, can be a powerful approach to deciphering protein structure and/or enzyme-substrate reaction mechanisms.
Typically, deletions or substitutions of amino acids are made at the gene or DNA level, by recombinant DNA techniques which rely on the use of restriction endonucleases. However, restriction endonucleases available have a limited array of target sites in DNA (usually palindromic hexanucleotide or octanucleotide sequences). Deletion of a particular in-frame trinucleotide or trinucleotides may not be possible because there may be no suitably located restriction sites. As a result, presently-available methods of altering an amino acid sequence by altering the DNA sequence which encodes it, are limited in their applicability.